Method and reagent for producing narrow, homogenous reagent strips

ABSTRACT

The present invention concerns a reagent coating mass which can be used in slot-die-coating of flat support materials in the manufacturing processes of test strips. Advantageously, the reagent mass of the invention exhibits certain superior rheological properties such as viscosity, surface tension and thixotropy. The reagent mass is preferably used to coat thin, narrow and homogeneous stripes of reagent material onto flat web material.

RELATED APPLICATIONS

This application is a divisional of application Ser. No. 10/871,966,filed Jun. 18, 2004, which claims the benefit of U.S. ProvisionalApplication No. 60/480,397, filed Jun. 20, 2003, which are herebyincorporated by reference.

This application is related to commonly assigned U.S. application Ser.No. 10/871,673 entitled “Reagent Stripe for Test Strip” (hereinafter“Reagent Stripe application”), filed on Jun. 18, 2004 (Attorney DocketNo. 007404-000566), which is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates generally to reagents used in biosensorsor test strips and more particularly to the production of narrow,homogenous reagent stripes on flat surfaces of test strips.

BACKGROUND AND SUMMARY

Of the numerous methods for applying reagents to test strips, in thepast electrochemical biosensors have mainly been produced by usingprinting techniques such as screen printing processes or dispensingtechniques for liquid reagent application and subsequent drying, (see.e.g., U.S. Pat. No. 5,437,999 and WO 97/02487). In connection withso-called “capillary fill” test strips, these dispensing methods havesuccessfully been employed, as in the production of Roche DiagnosticsAccuChek® Advantage test strips. While these techniques allow for theproduction of reliable electrochemical biosensors, they are not wellsuited for high throughput production lines. In addition, thesedispensing techniques suffer from the disadvantage of inhomogeneousdrying of the reagent, which leads to non-uniform reagent thickness overthe covered electrode area. Also, the above mentioned techniques are notsuited for the reliable and reproducible production of extremely thinreagent layers (10 μm or less). Therefore, there exists a need forimproved reagent application methods.

Blade coating of reagent compositions onto flat substrates has beensuggested and successfully been employed in the production of reagentfilms coated for example on transparent polymeric substrates (e.g., U.S.Pat. Nos. 5,437,999 and 6,036,919). Usually, films of a width of severalcentimeters to several meters can be produced by this method. For theproduction of test strips, the so created reagent layers are cut intosmall stripes and then applied to the test strip substrate. Bladecoating of reagent masses has the disadvantage that—although the centerportion of the film is homogenous in thickness—at the edge of the coatedarea inhomogeneities are found which are believed to be due to dryingeffects and edge effects. While these inhomogeneities are acceptable ifbroad bands of reagents are coated onto substrates since theinhomogeneous edge portions of the coating can be discarded by edgetrim, these inhomogeneities become more and more unacceptable as thereagent stripe to be coated becomes smaller/narrower.

WO 02/057781 discloses a method for manufacturing reagent strips fromweb material. Among other things, it discloses that the reagent materialmay be applied to the strip support material by laying down a narrowstripe of reagent material, which may or may not be supported by asupport carrier.

U.S. Patent Application Publication 2003/0097981, U.S. PatentPublication Number 2003/0099773, U.S. Pat. Nos. 6,676,995 and 6,689,411and EP 1 316 367) disclose a solution stripping system for laying downstripes of reagent solutions on a substrate. The system allowsslot-die-coating of reagent solutions to web material, e.g., forelectrochemical glucose sensors, which solutions have a low viscosity,from about 0.5 to 25 centipoises (cP=mPa-s).

U.S. Pat. Nos. 3,032,008; 3,886,898; and 4,106,437 teach coatingapparatuses useful for coating liquid material onto solid supports.

U.S. Pat. No. 6,036,919 discloses reagent films for optical bloodglucose test strips. The reagent composition comprises, among otherthings, a Xanthan gum.

U.S. Patent Application Publication Number 2003/0146113 disclosesreagent films for electrochemical coagulation sensors. The reagentcomposition comprises, among other things, carboxylated microcrystallinecellulose (Avicel® R591) as a film former.

None of the above-mentioned references satisfies the need for a reliablemethod for forming narrow (for example, less than 1 cm), thin (forexample, less than 10 μm) and homogeneous reagent stripes on solidsupport material for producing test strips, in particularelectrochemical test strips.

It is therefore an object of the invention to provide a method and acorresponding reagent composition with which extremely thin, narrow andhomogeneous reagent lines or stripes can be deposited onto flatsurfaces, for example, of web material and in particular onto theelectrode areas of electrochemical biosensor test strips.

This object is reached by the present invention concerning a reagent fora slot-die-coating process for narrow and homogenous reagent stripes.

In a first aspect, the present invention concerns a reagent compositionshowing shear thinning, slightly thixotropic or thixotropic behavior.

In a second aspect, the present invention concerns a method of coatingthe shear thinning, slightly thixotropic or thixotropic reagentcomposition onto web material using a slot-die-coating process.

In a further aspect, the present invention concerns analytical testelements comprising the shear thinning, slightly thixotropic orthixotropic reagent.

In still another aspect, the present invention concerns reagentcompositions that are shear thinning and at least slightly thixotropic.It also concerns analytic test elements and methods for making analytictest elements that include using shear thinning and at least slightlythixotropic reagent compositions.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows schematically in 6 steps (A-F) how an electrochemical testelement with a single reagent zone is manufactured using theslot-die-coating process of the present invention.

FIG. 2 shows schematically in 6 steps (A-F) how an electrochemical testelement with two reagent zones is manufactured using theslot-die-coating process of the present invention.

FIG. 3 shows the results of a profilometric measurement across thereagent stripe according to Example 1.

FIG. 4 represents the data of profilometric measurements across reagentstripes according to the present invention.

FIG. 5 represents the data of profilometric measurements across reagentstripes without the use of rheological modifiers of the presentinvention.

FIG. 6 is a photograph of a microscope view of a reagent stripe coatedonto a web material according to the present invention.

FIG. 7 shows two photographs (FIGS. 7A and 7B) of a microscope view of areagent stripe coated onto a web material without the use of rheologicalmodifiers of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

For the purposes of promoting an understanding of the principles of theinvention, reference will now be made to the specific embodimentsillustrated herein and specific language will be used to describe thesame. It will nevertheless be understood that no limitation of the scopeof the invention is thereby intended. Any alterations and furthermodifications in the described processes or devices and any furtherapplications of the principles of the invention as described herein, arecontemplated as would normally occur to one skilled in the art to whichthe invention relates. Preferred embodiments of the invention aresubject of the dependent claims.

The reagent composition of the present invention is shear thinning,slightly thixotropic or thixotropic. Thixotropic reagent compositionsare reagent compositions that show rheologic behavior depending onwhether or not external shear force is applied to the reagentcomposition. Shear thinning reagent compositions are reagentcompositions that become thinner, i.e., less viscous, when a shear forceis applied to them. In general, before applying a shear force to thereagent composition of the present invention, the composition has acertain viscosity. When a shear force is applied to the composition, itsviscosity is reduced. If viscosity increases again—with a certaintime-dependency—after the shear force is stopped, the reagentcomposition shall be regarded as being “shear thinning.” If viscosityincreases only with a certain delay after the shear force is stopped thereagent composition shall be regarded as being “thixotropic.”

Thixotropy is a special case of pseudoplasticity. The thixotropic fluidundergoes “shear thinning.” But as shear forces are reduced, viscosityrebuilds and increases at a slower rate, thus producing a hysteresisloop. Slightly thixotropic fluids have a less pronounced hysteresis. Inaddition, the thixotropic behavior is influenced considerably by theshear history of the material under investigation. In comparativemeasurements, care should be taken to ensure that an identical or atleast very similar history of the samples to be compared is given.

The reagent compositions of the present invention are useful inslot-die-coating processes. During slot-die-coating, the fluid reagentcomposition is applied to a solid substrate, preferably a substrate inthe form of a web material, by forcing the reagent liquid or slurrythrough the slot of a slot-die-coating head. Usually, the web materialpasses the slot at a certain distance with certain speed. However, it isalso possible that the slot-die-coating head moves across the webmaterial, or that the slot-die head and web both move.

To achieve the objects of the present inventions, it is advantageousthat the rheologic properties of the reagent composition used as acoating mass are within certain preferred ranges: The viscositypreferably is between about 70 and about 130 mPa-s, most preferably inthe range between 95 and 115 mPa-s. The surface tension rangesadvantageously between 30 and 50 mN/m and preferably is about 40±2 mN/m.It is also important that the coating mass shows shear thinning,slightly thixotropic or thixotropic behavior.

One aspect of the present invention is the inclusion of Xanthan gum intothe reagent coating mass. One brand of Xanthan gum that can be used isKeltrol®. This component shows an influence on the thixotropy of thereagent mass. Reagent coating masses containing Xanthan gum, forexample, Keltrol®, allow the production of extremely thin reagentlayers. Preferably, the reagent layer dried films have a thickness lessthan 10 μm, particularly preferred are dried reagent layers in the rangeof 1.5 to 5 μm thick.

It has turned out that the incorporation of silica into the reagentcompositions of the present invention has an advantageous effect for theviscosity and thixotropy behavior of the reagent. Both properties areenhanced by the addition of silica. Preferably, untreated, hydrophilicsilica is used. The particle size of a preferred form of silica rangesfrom about 1 to 7 μm. It has turned out that silica unexpectedlyenhances the thixotropic behavior of other components of the coatingmass, in particular of carboxymethyl cellulose and Keltrol®. Also,silica particles in the dry film prevent backside transfer between thecoated stripe and the backside of the web, allowing storage of thecoated web material as rolls of material. In addition, silica particlesin the dry film increase the specific surface of the reagent coating,enabling, for example, rapid dissolving of the reagent in a sampleliquid. In capillary fill biosensors comprising reagent stripesincluding the reagent composition of the present invention, silica alsoimproves capillary fill times and migration of components in the reagentstripe.

Yet another additive for the enhancement of viscosity and thixotropy ofthe reagent is carboxymethyl cellulose (CMC). Especially preferredembodiments of the inventive reagent composition therefore compriseXanthan gum, for example, Keltrol®, silica and CMC.

The reagent compositions of the present invention allow the formation ofthin reagent layers, for example, the production of electrochemicalbiosensors. Thin reagent layers have several advantages:

Sample components are in excess compared to the reagent components,therefore not limiting in the determination reactions.

Thin reagent layers can be made homogenous in thickness.

Thin reagent layers contain only small amounts of reagent, which in turnlead to fast reaction times.

The reactions only have short diffusion times.

The thin reagent layers are quickly soluble and therefore lead to quickreagent availability and a rapid equilibration of the matrix aftersample rehydration of the reagent stripe, which in turn leads to fastmeasurements.

The inventive reagent layers can not only be made very thin but alsoshow a high homogeneity down web and across web in the reaction area.The reagent layer in the test area is flat and uniform in thickness.Thickness variations in the coated stripe occur preferably only on theouter 0.2 cm (or less) edges of the stripe. In preferred embodiments,these areas advantageously can either be covered during sensor assemblyby spacer layers or can be trimmed from the completed sensor in thefinal assembly process.

Apart from the above-mentioned components, which influence the rheologicproperties of the reagent composition of the present invention, thereagent may further comprise one or more substances (ingredients) of thefollowing substance classes. Substances, additives and ingredients thatmay be added to the reagent includes, but are not limited to, thefollowing:

buffers, for example, phosphate buffers;

enzymes, such as, glucose dehydrogenase, glucose dye oxidoreductase,glucose oxidase and other oxidases or dehydrogenases such as for lactateor cholesterol determination, esterases etc.;

mediators such as nitrosoanilines, ferricyanide, ruthenium hexamine,osmium complexes;

stabilizers, such as trehalose, sodium succinate;

thickeners, such as Keltrol®, CMC

proteins, such as enzymes, bovine serum albumin

indicators;

dyes;

surfactants, such as Mega 8®, Geropon®; Triton®, Tween®, Mega 9®, DONS;

film formers, such as Keltrol®, Propiofan®, polyvinyl pyrrolidone,polyvinyl alcohol, Klucel®;

co-factors for enzymes, such as NAD, NADH, PQQ; and

silica, for example, DS 300, DS 320, milled silica of DS 300, milledsilica of DS 320.

Non-limiting examples of enzymes and mediators that may be used inmeasuring particular analytes are listed below in Table 1.

TABLE 1 A partial list of some analytes, enzymes and mediators that canbe used to measure the levels of particular analytes. Mediator AnalyteEnzymes (Oxidized Form) Additional Mediator Glucose GlucoseDehydrogenase Ferricyanide and Diaphorase Glucose Glucose-DehydrogenaseFerricyanide (Quinoprotein) Cholesterol Cholesterol Esterase andFerricyanide 2,6-Dimethyl-1,4- Cholesterol Oxidase Benzoquinone2,5-Dichloro-1,4- Benzoquinone or Phenazine Ethosulfate HDL CholesterolEsterase and Ferricyanide 2,6-Dimethyl-1,4- Cholesterol CholesterolOxidase Benzoquinone 2,5-Dichloro-1,4- Benzoquinone or PhenazineEthosulfate Triglycerides Lipoprotein Lipase, Ferricyanide or PhenazineMethosulfate Glycerol Kinase and Phenazine Glycerol-3-PhosphateEthosulfate Oxidase Lactate Lactate Oxidase Ferricyanide2,6-Dichloro-1,4- Benzoquinone Lactate Lactate DehydrogenaseFerricyanide and Diaphorase Phenazine Ethosulfate, or PhenazineMethosulfate Lactate Diaphorase Ferricyanide Phenazine Ethosulfate, orDehydrogenase Phenazine Methosulfate Pyruvate Pyruvate OxidaseFerricyanide Alcohol Alcohol Oxidase Phenylenediamine BilirubinBilirubin Oxidase 1-Methoxy- Phenazine Methosulfate Uric Acid UricaseFerricyanideIn some of the examples shown in Table 1, at least one additional enzymeis used as a reaction catalyst. Also, some of the examples shown inTable 1 may utilize an additional mediator, which facilitates electrontransfer to the oxidised form of the mediator. The additional mediatormay be provided to the reagent in lesser amount than the oxidized formof the mediator. While the above assays are described, it iscontemplated that current, charge, impedance, conductance, potential, orother electrochemically indicated property of the sample might beaccurately correlated to the concentration of the analyte in the samplewith an electrochemical biosensor in accordance with this disclosure.

Examples of reagent compositions are given as Examples 1, 2, 3 and 4 forelectrochemical blood glucose and coagulation sensors, respectively.

In a preferred embodiment, the above reagent compositions are applied tosubstrates which already contain the electrode traces or circuits of anelectrochemical sensor by means of a slot-die-coating process. Anexample of this process is given in Example 5.

The preferred fabrication technique for these electrode circuits uses alaser ablation process. For a further discussion of laser ablation,please see WO 01/25775, which is hereby incorporated by reference in itsentirety. Most preferably, the technique uses a reel-to-reel laserablation process. This process can be used in reel-to-reel fashion toform extremely thin metal structures on polymeric substrates, whichmetal structures can be used as electrode traces in electrochemicalsensors. The reagent can be applied to these structures using the aboveprocess.

Surprisingly, it has been found that the capillary channel and spacerstructure of the sensor can be formed by using a double sided adhesivetape with a respective cutout as a spacer structure and covering partsof the reagent layer on the electrode substrate. Unexpectedly, noleakage of sample liquid can be observed at the positions where thedouble-sided adhesive tape covers the reagent film. Therefore, it ispossible to first make structured electrode traces by a laser ablationprocess on a web material, then slot-die-coat the reagent material andsubsequently define the active reagent area which comes into contactwith the blood sample by using a respectively formed double sidedadhesive spacer. This process can advantageously be used to eliminatetolerances in the production line. Especially, masking the reagentcoating with the spacer can be used to precisely define the actualreaction area.

In the second aspect of the present invention, the invention concerns amethod or process for producing a reagent layer on a solid supportmaterial using the shear thinning, slightly thixotropic or thixotropicreagent composition of the invention. The process includes providing asolid support material such as a web of plastics material like Melinex®329 of DuPont. During the process of the present invention, the solidsupport material is moved relative to a slot-die-coating head. Usually,the solid support web material is transported in a reel-to-reel processacross the slot of the die-coating head. However, it is also possible,to move the die-coating head and keep the web material stationary.During the movement of the web material relative to the die-coatinghead, a defined distance between the web and the die-coating head ismaintained. Preferably, the coating gap is in the range of between 30and 90 μm, typically between 68 and 83 μm, most preferred between 72 and76 μm. By forcing the reagent composition through the slot of theslot-die-coating head, the reagent is deposited onto the solid supportmaterial, forming a continuous stripe of reagent on the solid supportmaterial. As mentioned above, the web material may comprise electrodetraces and the reagent stripe may partly cover these traces. Preferably,in the dried state the reagent stripe has a width of less than 1 cm anda height of less than 10 μm.

Preferably, the solid support material is moved relative to theslot-die-coating head at a speed of between 20 and 80 m/min, mostpreferably at a speed of between 30 and 40 m/min.

Preferably, the reagent composition is delivered to the solid supportmaterial at a coating flux of 5.5 to 30 g/min, most preferably at a fluxof 13 to 15 g/min.

Subsequently, the deposited reagent stripe is dried either under ambientconditions or in a heated airflow.

In a further aspect, the invention concerns analytical test elementsthat comprise the above reagent composition. Preferably, the analyticaltest elements of the present invention are manufactured according to theprocess as described above.

The invention has the following advantages:

1. Sensors requiring small sample volumes (typically 100 to 1000 nl) caneasily be constructed using the slot die coated dry film andspacer/capillary channel lamination processes. The dry film stripe is ofuniform thickness and is homogeneous over the electrochemical reactionarea. The required capillary dimensions/imprecision of the sensor isdependent on the variation in spacer thickness and the construction ofthe capillary channel.

2. The slot-die-coating technology can be paired with a sophisticatedlayout of the electrodes design, thus enabling the capability ofminiaturizing and creating multiple applications in the sensorcapillary, (for example, staggering two or more lines/stripes ofdifferent reagents within an adequately designed layout of electrodes).Two staggered slot dies or a special slot die assembly designed for twoor more fluids can be used to achieve this goal. The coating fluidspreferably will have properly matching rheologic properties. The besttechnological case is achieved if the coating windows of the differentfluids have a consistent overlapping region.

3. The slot-die-coating film application technology paired and combinedwith the rheologic properties of the reagent enables homogeneouscoatings using a reel to reel coating process for rapid production ofdiagnostic sensors.

4. Thixotropy or shear thinning behavior is the main rheologic featureof the fluid to be coated in respect to the mass distribution and itsprofile across the coated layer, impacting on the flatness,repeatability and homogeneity of the wet and dried layer. This featureis reached by using Xanthan gum, for example, Keltrol®, CMC and Silicain a concentration and combination to match the desired shear thinning,slightly thixotropic or thixotropic behavior of the coating fluid.

Surprisingly, it has been found that the role of silica, in particularthe preferred untreated, hydrophilic silica, preferably with a particlesize D50 (i.e., 50% of the particles have a size of the given size orbelow) of 1 to 7 μm, in the “wet” status (in the coating fluid) is thatin combination with the film thickeners (Keltrol® and CMC, either one orboth of them) silica increases the viscosity and enhances the shearthinning, slightly thixotropic or thixotropic behavior of the coatingfluid.

Silica acts in the dried state to, among other things:

a) prevent back transfer of the dried film on the un-coated side of thefoil/carrier if the web material is wound to rolls after the coating anddrying processes, and

b) enlarge the specific surface of the dried coating layer as comparedto a smooth coating layer. Without wishing to be tied to any specifictheory, this is likely due to the particle-size distribution of silicaparticles. Since the speed of fluid transport is increased by the ratiobetween the surface area and the fluid volume, this enlarged specificsurface is speeding up the wetting process of the dried film and inconsequence leads to a shorter capillary fill time.

The present invention is further elucidated by the following Examplesand Figures. With respect to the Figures whenever possible like numbers,letters and symbols refer to like structures, features and elements. Forexample, unless otherwise stated in the application the following keyapplies:

1 indicates a web;

2 indicates a sputtered metal film;

3 indicates a working electrode of electrode pair 1;

3′ indicates a working electrode of electrode pair 2;

4 indicates a reference/counter electrode of electrode pair 1;

4′ indicates a reference/counter electrode of electrode pair 2;

5 indicates a reagent stripe 1;

5′ indicates a reagent stripe 2;

6 indicates a spacer (e.g., double sided adhesive);

7 indicates a top foil; and

8 indicates a vent opening in top foil.

FIGS. 1 and 2 are schematic representations of the several steps thatare done during the manufacturing process for electrochemical testelements using the reagent composition and process of the presentinvention. A person of ordinary skill in the art would readily recognizethat the process can be used with other electrode configurations andwith multiple stripes having the same or different composition anddifferent positions on the strip. It is to be noted that the processesdescribed in FIGS. 1 and 2 could also be carried out without electrodespresent on the test strips. A person of ordinary skill in the art wouldreadily recognize that the process and reagent described herein can alsobe adapted to optical test elements as well.

Parts A and B of FIGS. 1 and 2 are identical and show a polymer web (1),preferably an inert plastic material such as Melinex® 329 of DuPont(see, e.g., part A), on which is coated a metal layer (2) (see, e.g.,part B) by conventional techniques such as sputtering, chemical,electrochemical or physical vapor deposition, etc.

Preferably, the metal layer (2) subsequently is structured by forexample a laser ablation process. This process removes parts of themetal layer (2) and discrete structures of metal which can act aselectrodes (3, 4) remain on the surface of the polymer web (1). Itshould be understood, however, that conventional printing techniques orlithographic processes can also be used to create electrodes (3, 4) onthe polymer web (1).

After the laser ablation step in FIG. 1, part C, two electrodes (3, 4)are formed on the polymer web. In FIG. 2, part C, two pairs ofelectrodes (3, 3′, 4, 4′) are formed.

In the next step (shown in part D of FIGS. 1 and 2), reagent stripes 5,5′ are deposited over the active area of the working and counterelectrodes (3, 3′, 4, 4′). The reagent composition is applied on theelectrode structure by the slot-die-coating process of the presentinvention.

In part E of FIGS. 1 and 2, a spacer layer (6) is laminated to theelectrode structure of part D of FIGS. 1 and 2. The spacer (6) ispreferably a double-sided adhesive tape that covers all parts of thereagent and electrode structures that are not to be brought into contactwith liquid sample. In addition, the spacer (6) has a cutout thatdefines the reactive area of the reagent and the underlying electrodes.At the opposite end of the spacer (6), i.e., the end where the electrodeleads are located that are not covered by reagent composition, thespacer (6) leaves free parts of the electrode structures that can beused to connect the test strip to a respective test strip reading meter.

Spacer (6) preferably covers a narrow part (less than 2 mm) of thereagent (5 in FIG. 1, 5′ in FIG. 2) to mask eventual inhomogeneous edgeregions of the reagent coating.

After laminating the spacer (6) to the electrode and reagent web, in apreferred embodiment, part of the web material is cut off to trim thereagent stripe (5).

In part F of FIGS. 1 and 2, a top foil (7), preferably an inert plasticscover, is placed onto the surface of the spacer (6) that is not incontact with the polymer web (1). The polymer web (1), the spacer (6)and the top foil (7) form a 3D capillary channel which is defined by thethickness of the spacer (6) and the dimensions of the cut-out in thespacer. To enable a filling of the capillary space, preferably eitherthe top foil (7) or the polymer web (1) has a vent opening (8).

As is clear for those skilled in the art, the surfaces of either thepolymer web (1) or the top foil (7) that face the capillary space can berendered hydrophilic by a respective hydrophilic treatment, for example,by coating with a surfactant or plasma treatment.

EXAMPLES

The following Examples provided by way of illustration and not by way oflimitation, will disclose more details of the invention:

Example 1 Reagent Composition for Use in an Electrochemical AmperometricGlucose Biosensor

An aqueous mixture of the following components was prepared:

Substance Source % w/w Keltrol ® F (Xanthan gum) Kelco 0.2136%Carboxymethyl cellulose (CMC) Hercules-Aqualon 0.5613%Polyvinylpyrrolidone (PVP) K25 BASF 1.8952% Propiofan ®(polyvinylchloride) BASF 2.8566% (50% water) Glucose-dye-oxidoreductaseRoche Diagnostics 0.3310% (GlucDOR) (E.C. 1.1.99.17) pyrroloquinolinequinine (PQQ) Roche Diagnostics 0.0092% Sipernat ® 320 DS Degussa Hüls2.0039% (synthetic, amorphous precipitated silica) Na-Succinat × 6 H₂OMallinckrodt 0.4803% Chmeicals Trehalose Sigma-Aldrich 0.4808% KH₂PO₄ J.T. Baker 0.4814% K₂HPO₄ J. T. Baker 1.1166%N,N-Bis-(hydroxyethyl)-3-methoxy-p- Roche 0.6924% nitroso anilineDiagnostics Mega 8 ® (n-Octanoyl- Dojindo 0.2806% N-methylglucamide)Geropon ® T 77 (Sodium Rhodia Chimie 0.0298% N-methyl N-oleyl taurate)KOH Merck 0.1428% Water, double distilled 89.9558%

The reagent matrix was custom modified to meet the demands of theslot-die-coating process. Silica, Keltrol® (Xanthan Gum), carboxymethylcellulose (CMC) and surfactants were added to the coating matrix tomodify the rheology of the reagent mass. Surfactant concentrations wereadjusted to obtain surface tensions (measured with a Tensiometer K10T(Kruess)) in the most preferred range of 33 to 42 mN/m. Surface tensionin this range promotes better adhesion and controlled spreading of thecoated stripe on the web. The most preferred viscosity range measuredusing a Rheomat 115 (Contraves) for the coating mass is 95 to 115 mPa-s.The polymers and the silica also impart thixotropic behavior to thecoating. Coatings shear thin as they are dispensed through the slot diehead onto the web. This reduces the apparent viscosity of the coating.

Stripes of reagent coating mass with these lower viscosities show amigration of the stripe edges and reagent components toward the centerof the stripe during the drying process. This migration leads to anirregular and irreproducible surface profile in the middle of the driedstripe. Dispense of coatings having shear thinning, slightly thixotropicor thixotropic properties show the same shear thinning effects. However,the viscosity of the coated stripe returns to near the apparentviscosity shortly after being dispensed and before entering the dryingregion. The migration of the stripe edges towards the center duringdrying is retarded. As illustrated in FIG. 3, this leads to a flatreproducible region in the center of the stripe, in the reaction area.Thinner films further retard the migration of the coating edges to thecenter of the coated stripe.

Example 2 Reagent Composition for an Electrochemical AmperometricCoagulation Sensor

An aqueous mixture of the following components was prepared:

End Concentration Substance Source in Reagent Glycine Sigma 23 g/lPolyethylenglycol Sigma 23 g/l Sucrose Sigma 55 g/l Bovine Serum AlbuminSigma 6.9 g/l Mega 8 ® (n-Octanoyl-N- Dojindo 1 g/l methylglucamide)Resazurin Sigma-Aldrich 1.4 g/l Chemie GmbH 5.6 mmol/l Polybrene ®(hexadimethrine Sigma 0.015 g/l bromide) Moviol ® 4/86 (poly vinylClariant GmbH 20 g/l alcohol) Keltrol ® F (Xanthan gum) Kelco 2.89 g/lElectrozym TH Roche Diagnostics 1.226 g/l (reduced Chromozym TH; 1.9mmol/l reduced tosyl-glycyl-prolyl- arginine-4-nitranilide acetate) soybean phospholipids solution of recombinant tissue Dade-Behring 109 μg/lfactor

Example 3 Alternative Reagent Composition for an ElectrochemicalAmperometric Glucose Biosensor

An aqueous mixture of the following components was prepared:

Substance Source % w/w Keltrol ® F (Xanthan gum) Kelco 0.20% Gantrez ®S97 (Methyl ISP 2.48    vinylether/maleic anhydride copolymer)Polyvinylpyrrolidone (PVP) K25 BASF 1.93% Propiofan ®(polyvinylchloride) BASF 2.94% (50% water) Glucose-dye-oxidoreductaseRoche Diagnostics 0.33% (GlucDOR) (E.C. 1.1.99.17?) pyrroloquinolinequinine (PQQ) Roche Diagnostics 0.0093%  Silica FK 300 DS Degussa Hüls1.77% KH₂PO₄ J. T. Baker 0.48% K₂HPO₄ J. T. Baker 1.47%N,N-Bis-(hydroxyethyl)-3-methoxy- Roche Diagnostics 0.69% p-nitrosoaniline Mega 8 ® (n-Octanoyl- Dojindo 0.29% N-methylglucamide) Geropon ®T 77 (Sodium Rhodia Chimie 0.030%  N-methyl N-oleyl taurate) KOH Merck1.14% Water, double distilled 86.227% 

Example 4 Alternative Reagent Composition for an ElectrochemicalAmperometric Glucose Biosensor

An aqueous mixture of the following components was prepared:

Substance Source % w/w Keltrol ® F (Xanthan gum) Kelco 0.20% Gantrez ®S97 (Methyl ISP 0.50% vinylether/maleic anhydride copolymer)Carboxymethyl cellulose (CMC) Hercules-Aqualon 0.50%Polyvinylpyrrolidone (PVP) K25 BASF 1.90% Propiofan ®(polyvinylchloride) BASF 2.89% (50% water) Glucose-dye-oxidoreductaseRoche Diagnostics 0.34% (GlucDOR) (E.C. 1.1.99.17?) pyrroloquinolinequinine (PQQ) Roche Diagnostics 0.0093%  KH₂PO₄ J. T. Baker 0.48% K₂HPO₄J. T. Baker 1.46% N,N-Bis-(hydroxyethyl)-3-methoxy- Roche Diagnostics0.71% p-nitroso aniline Mega 8 ® (n-Octanoyl-N- Dojindo 0.28%methylglucamide) Geropon ® T 77 (Sodium Rhodia Chimie 0.030%  N-methylN-oleyl taurate) KOH Merck 0.31% Water, double distilled 90.384% 

Example 5 Coating Process

The polymer web (Melinex® 329, DuPont) is moved into the coating area,containing a slot die head and a back up roller. The slot die head (TSE,Switzerland) is zeroed to the web surface and adjusted to a slot to webgap of 74 μm. Web speed is ramped up from 0 to 38 m/min for depositionof coating on the web. The reagent matrix can be delivered to the slotdie head using a variety of means including gear pumps, pistons,syringes, bladder systems. The reagent delivery system is adjusted to awater flow of 13.58 ml/min to deliver a coat weight of 53 g/m² throughthe coating head. The width of the resulting coated stripe is 7.0±0.3mm. The coating is dried in the heated drying zone (length 15 m,temperature 110° C., at a speed of 38 m/min) and rewound on spools atthe rewind station.

FIGS. 3 to 5 show the results of profilometric measurements across thereagent stripe according to this example. The profilometer system usedwas a Dektak IIA Surface Profile Measuring System (Veeco InstrumentsInc., Sloan Technology Division, Dallas, Tex.). Profile data from theDektak IIA were baseline corrected.

In FIG. 3, P (mm) denotes the x-position of the scan across the web andreagent stripe (in mm) and H (μm) denotes the respective relative heightof the coating (in μm). The reagent mass was prepared according toExample 1. As can be seen, the reagent stripe has a cross-sectionalwidth of about 7 mm and a respective average center height ofapproximately 5 μm. The edges of the reagent coating are relativelysharp. The homogeneous plateau region of the coating fills approximately80% of the reagent stripe width.

The profile of the reagent coating as depicted in FIG. 3 is typical forcoatings according to the present invention. For reagent stripes of 10mm or less in width, sharp edges can be obtained, which ramp up from theunderlying web material (corresponding to a coating height of zero) tothe plateau region in the center of the coating within 1 mm on each sideor less (i.e. 80% or more of the coating belong to the homogeneouscenter plateau region). Within the center region, the reagent coating ispractically uniform in thickness.

FIG. 4 shows the results of profilometric measurements across thereagent stripe prepared according to this example. Scan Distance (μm)denotes the x-position of the scan across the web and reagent stripe (inμm) and Height (μm) denotes the respective relative height of thecoating (in μm). The reagent mass was prepared according to Example 1with milled silica. FIGS. 4A to 4E give the results for coating weightsof 20, 25, 30, 40 and 50 mg/m², respectively.

FIG. 5 illustrates the results of profilometric measurements across areagent stripe prepared according to a comparative example, i.e., not inaccordance with the teachings of the present invention. Scan Distance(μm) denotes the x-position of the scan across the web and reagentstripe (in μm) and Height (μm) denotes the respective relative height ofthe coating (in μm). The reagent mass was prepared according to Example1 however without the presence of the rheological modifiers Keltrol®,CMC and silica.

FIGS. 5A and 5B show that without rheological modifiers the driedreagent coating tends to form inhomogeneous reagent stripes on the webmaterial. In FIG. 5A, the reagent concentrates in the center portion ofthe coated stripe; in FIG. 5B, the reagent concentrates in two regionslocated between the center and the edge portions of the reagent stripes.In both cases, the edge portions are depleted from reagent.

Comparison of FIG. 5 (comparative example) with FIGS. 3 and 4 (bothaccording to the present invention) reveals the advantageous effects ofthe reagent composition and process of the present invention.

FIG. 6 is a photograph of a microscope view of a reagent stripe (centraldark rectangular area) coated onto a web material (light areas aroundthe central stripe) from a reagent composition according to Example 1(and comparable to the profilometric data shown in FIG. 4). Coating wasdone according to Example 5. The coated stripe shows good homogeneityacross the coating direction (coating direction was from top to bottom)as well as along this direction.

In stark contrast to the smooth and uniform reagent layer shown in FIG.6, FIGS. 7A and 7B are photographs of microscope views (comparativeexamples) of reagent stripes coated onto web material with profilometricdata comparable to that shown in FIG. 5. Coating was done according toExample 5; however, the reagent did not contain the rheologicalmodifiers. The coated stripes clearly show inhomogeneities across thecoating direction (coating direction was from top to bottom). Forexample, regions of thicker reagent are manifested by the dark bandsrunning along the stripes. FIG. 7A shows one such dark band positionedin about the middle of the stripe (compare FIG. 5A), whereas FIG. 7Bshows two dark bands (compare FIG. 5B). These one or two regions ofthicker reagent coatings (dark zones) within the reagent stripe arebelieved due to drying effects of the reagent coating materials.

Example 6 Variation of Rheological Modifiers in the Reagent Compositionof Example 1

In the reagent composition of Example 1 the contents of the ingredientsCMC, Keltrol®, Propiofan® and PVP were varied in accordance with thefollowing Table. Ingredient contents are given in % w/w and viscosity isgiven in mPa-s.

CMC Keltrol ® PVP Propiofan ® Silica Viscosity Thixotrophic 0.56 0.211.9 2.86 2 117 yes 0.476 0.28 1.52 2.29 2 99 yes 0 0.77 1.52 2.29 2 69.5yes 0.77 0 2.28 3.43 2 149 yes 0.504 0.4 1.9 2.86 2 123 weak

All publications, patents and patent applications cited in thisspecification are herein incorporated by reference, as if eachindividual publication, patent, or patent application was specificallyand individually indicated to be incorporated by reference and set forthin its entirety herein.

While preferred embodiments incorporating the principles of the presentinvention have been disclosed hereinabove, the present invention is notlimited to the disclosed embodiments. Instead, this application isintended to cover such departures from the present disclosure as comewithin known or customary practice in the art to which this inventionpertains and which fall within the limits of the appended claims.

1. A process for producing a reagent layer on a solid support material,comprising the steps of: providing a solid support material; moving saidsolid support material relative to a slot-die-coating head; maintaininga defined distance between the surface of said solid support materialand said slot-die-coating head; depositing a shear thinning, at leastslightly thixotropic reagent through said slot-die-coating head tocreate a continuous stripe of reagent on said solid support material,wherein said reagent has a viscosity of between about 70 and 130 mPa-sand a surface tension of between about 30 and 50 mN/m; and drying thecoated reagent stripe on said support material.
 2. The process accordingto claim 1, wherein said reagent is deposited on said solid support at acoating flux rate of about 5.5 to about 30 g/min.
 3. The processaccording to claim 1, wherein said reagent is deposited on said solidsupport at a coating flux rate of about 13 to about 15 g/min.
 4. Theprocess according to claim 1, wherein said defined distance between saidslot-die-coating head and said support material is between 30 and 90 μm.5. The process according to claim 1, wherein said support material movesrelative to said slot-die-coating head at a rate of between 20 and 80m/min.
 6. The process according to claim 1, wherein said supportmaterial moves relative to said slot-die-coating head at a rate ofbetween 30 and 40 m/min.
 7. The process according to claim 1, whereinsaid stripe of reagent is less than 1 cm wide.
 8. The process accordingto claim 1, wherein after drying, said reagent on said solid supportmaterial has a height of less than 10 μm.
 9. The process according toclaim 1, said reagent including at least one of buffers, enzymes,mediators, stabilizers, thickeners, proteins, indicators, dyes, filmformers, surfactants and co-factors.
 10. The process according to claim9, said reagent further including at least one of silica, xanthan gumand CMC.
 11. The process according to claim 9, said reagent furtherincluding silica and at least one of xanthan gum and CMC.
 12. Theprocess according to claim 9, said reagent further including silica,xanthan gum and CMC.
 13. In a process for preparing biosensors from awebbing of a substrate, the improvement comprising: depositing acontinuous stripe of reagent onto the webbing in the form of a shearthinning, at least slightly thixotropic reagent having a viscosity ofbetween about 70 and 130 mPa-s and a surface tension of between about 30and 50 mN/m; and drying the coated reagent stripe on said supportmaterial.
 14. A process for preparing biosensors comprising: depositinga continuous stripe of reagent onto a webbing in the form of a shearthinning, at least slightly thixotropic reagent having a viscosity ofbetween about 70 and 130 mPa-s and a surface tension of between about 30and 50 mN/m; applying a spacer layer over the reagent; and attaching thespacer layer to the webbing.